Efficacy of anti-HLA-DR antiboddy drug conjugate IMMU-140 (hL243-CL2A-SN-38) in HLA-DR positive cancers

ABSTRACT

The present invention relates to therapeutic immunoconjugates comprising SN-38 attached to an anti-HLA-DR antibody or antigen-binding antibody fragment. The immunoconjugate may be administered at a dosage of between 3 mg/kg and 18 mg/kg, preferably 4, 6, 8, 9, 10, 12, 16 or 18 mg/kg, more preferably 8, 10 or 12 mg/kg. When administered at specified dosages and schedules, the immunoconjugate can reduce solid tumors in size, reduce or eliminate metastases and is effective to treat cancers resistant to standard therapies, such as radiation therapy, chemotherapy or immunotherapy. The methods and compositions are particularly useful for treating AML, ALL or multiple myeloma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/281,453, filed Sep. 30, 2016, which was a divisional of U.S.patent application Ser. No. 14/667,982 (now U.S. Pat. No. 9,493,573),filed Mar. 25, 2015, which was a divisional of U.S. patent applicationSer. No. 13/948,732 (now U.S. Pat. No. 9,028,833), filed Jul. 23, 2013,which claimed the benefit under 35 U.S.C. 119(e) of Provisional U.S.Patent Application Ser. Nos. 61/749,548, filed Jan. 7, 2013, and61/736,684, filed Dec. 13, 2012. This application is acontinuation-in-part of U.S. patent application Ser. No. 15/484,308,filed Apr. 11, 2017, which claimed the benefit under 35 U.S.C. 119(e) ofProvisional U.S. Patent Application Ser. No. 62/373,591, filed Aug. 11,2016, and 62/322,441, filed Apr. 14, 2016. This application claims thebenefit under 35 U.S.C. 119(e) of Provisional U.S. Patent ApplicationSer. No. 62/373,591, filed Aug. 11, 2016, and 62/428,231, filed Nov. 30,2016. The text of each priority application incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 25, 2017, isnamed IMM369US1_SL.txt and is 9,851 bytes in size.

FIELD OF THE INVENTION

The present invention relates to therapeutic use of immunoconjugates ofantibodies or antigen-binding antibody fragments and camptothecins, suchas SN-38, with improved ability to target various cancer cells in humansubjects. Preferably, the antibodies or fragments are anti-HLA-DRantibodies or fragments, such as hL243. In other preferred embodiments,the antibodies and therapeutic moieties are linked via anintracellularly-cleavable linkage that increases therapeutic efficacy.In more preferred embodiments, the immunoconjugates are administered atspecific dosages and/or specific schedules of administration thatoptimize the therapeutic effect. The optimized dosages and schedules ofadministration of SN-38-conjugated anti-HLA-DR antibodies for humantherapeutic use disclosed herein show unexpected superior efficacy thatcould not have been predicted from animal model studies, allowingeffective treatment of cancers that are resistant to standardanti-cancer therapies, including the parental compound, irinotecan(CPT-11). The immunoconjugates may be administered alone or incombination with one or more other therapeutic agents, administeredbefore, concurrently with, or after the immunoconjugate. Exemplarytherapeutic agents that may be used in combination with anti-HLA-DRinclude, but are not limited to proteosome inhibitors such asbortezomib, Bruton kinase inhibitors such as ibrutinib, orphosphoinositide-3-kinase inhibitors such as idelalisib. In preferredembodiments, the cancer to be treated is an HLA-DR positive cancer, suchas B-cell lymphoma, B-cell leukemia, skin, esophageal, stomach, colon,rectal, pancreatic, lung, breast, ovarian, bladder, endometrial,cervical, testicular, melanoma, kidney, or liver cancer. Morepreferably, the cancer is AML (acute myelocytic leukemia), ALL (acutelymphocytic leukemia) or MM (multiple myeloma). Most preferably, thepatient to be treated has relapsed from or shown resistance to at leastone standard anti-cancer therapy, prior to treatment with theimmunoconjugate. However, the person of ordinary skill will realize thatin some embodiments the immunoconjugate may be employed in a front-linetherapy.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer in humans, andvirtually no application in other diseases, such as autoimmune diseases.The toxic agent is most commonly a chemotherapeutic drug, althoughparticle-emitting radionuclides, or bacterial or plant toxins, have alsobeen conjugated to MAbs, especially for the therapy of cancer (Sharkeyand Goldenberg, C A Cancer J Clin. 2006 July-August; 56(4):226-243) and,more recently, with radioimmunoconjugates for the preclinical therapy ofcertain infectious diseases (Dadachova and Casadevall, Q J Nucl Med MolImaging 2006; 50(3): 193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using verywell-defined conjugation chemistries, often at specific sites remotefrom the MAbs' antigen binding regions; (c) MAb-chemotherapeutic drugconjugates can be made more reproducibly and usually with lessimmunogenicity than chemical conjugates involving MAbs and bacterial orplant toxins, and as such are more amenable to commercial developmentand regulatory approval; and (d) the MAb-chemotherapeutic drugconjugates are orders of magnitude less toxic systemically thanradionuclide MAb conjugates, particularly to the radiation-sensitivebone marrow.

Camptothecin (CPT) and its derivatives are a class of potent antitumoragents. Irinotecan (also referred to as CPT-11) and topotecan are CPTanalogs that are approved cancer therapeutics (Iyer and Ratain, CancerChemother. Phamacol. 42: S31-S43 (1998)). CPTs act by inhibitingtopoisomerase I enzyme by stabilizing topoisomerase I-DNA complex (Liu,et al. in The Camptothecins: Unfolding Their Anticancer Potential, LiehrJ. G., Giovanella, B. C. and Verschraegen (eds), NY Acad Sci., NY922:1-10 (2000)). CPTs present specific issues in the preparation ofconjugates. One issue is the insolubility of most CPT derivatives inaqueous buffers. Second, CPTs provide specific challenges for structuralmodification for conjugating to macromolecules. For instance, CPT itselfcontains only a tertiary hydroxyl group in ring-E. The hydroxylfunctional group in the case of CPT must be coupled to a linker suitablefor subsequent protein conjugation; and in potent CPT derivatives, suchas SN-38, the active metabolite of the chemotherapeutic CPT-11, andother C-10-hydroxyl-containing derivatives such as topotecan and10-hydroxy-CPT, the presence of a phenolic hydroxyl at the C-10 positioncomplicates the necessary C-20-hydroxyl derivatization. Third, thelability under physiological conditions of the δ-lactone moiety of theE-ring of camptothecins results in greatly reduced antitumor potency.Therefore, the conjugation protocol is performed such that it is carriedout at a pH of 7 or lower to avoid the lactone ring opening. However,conjugation of a bifunctional CPT possessing an amine-reactive groupsuch as an active ester would typically require a pH of 8 or greater.Fourth, an intracellularly-cleavable moiety preferably is incorporatedin the linker/spacer connecting the CPTs and the antibodies or otherbinding moieties.

The human leukocyte antigen-DR (HLA-DR) is one of three isotypes of themajor histocompatibilty complex (MHC) class II antigens. HLA-DR ishighly expressed on a variety of hematologic malignancies and has beenactively pursued for antibody-based lymphoma therapy (Brown et al.,2001, Clin Lymphoma 2:188-90; DeNardo et al., 2005, Clin Cancer Res11:7075s-9s; Stein et al., 2006, Blood 108:2736-44). The human HLA-DRantigen is expressed in non-Hodgkin lymphoma (NHL), chronic lymphocyticleukemia (CLL), and other B-cell malignancies at significantly higherlevels than typical B-cell markers, including CD20. Preliminary studiesindicate that anti-HLA-DR mAbs are markedly more potent than other nakedmAbs of current clinical interest in in vitro and in vivo experiments inlymphomas, leukemias, and multiple myeloma (Stein et al., unpublishedresults).

HLA-DR is also expressed on a subset of normal immune cells, including Bcells, monocytes/macrophages, Langerhans cells, dendritic cells, andactivated T cells (Dechant et al., 2003, Semin Oncol 30:465-75). Thus,it is perhaps not surprising that prior attempts to develop anti-HLA-DRantibodies have been hampered by toxicity, notably infusion-relatedtoxicities that are likely related to complement activation (Lin et al,2009, Leuk Lymphoma 50:1958-63; Shi et al., 2002, Leuk Lymphoma43:1303-12).

The L243 antibody (hereafter mL243) is a murine IgG2a anti-HLA-DRantibody. This antibody may be of potential use in the treatment ofdiseases such as autoimmune disease or cancer, particularly leukemias orlymphomas, by targeting the D region of HLA. mL243 demonstrates potentsuppression of in vitro immune function and is monomorphic for allHLA-DR proteins. However, problems exist with the administration ofmouse antibodies to human patients, such as the induction of a humananti-mouse antibody (HAMA) response. A need exists for more effectivecompositions and methods of use of anti-HLA-DR antibodies, with improvedefficacy and decreased toxicity. A further need exists for moreeffective methods of preparing and administering antibody-CPTconjugates, such as anti-HLA-DR-SN-38 conjugates. Preferably, themethods comprise optimized dosing and administration schedules thatmaximize efficacy and minimize toxicity of the antibody-CPT conjugatesfor therapeutic use in human patients.

SUMMARY OF THE INVENTION

As used herein, the abbreviation “CPT” may refer to camptothecin or anyof its derivatives, such as SN-38, unless expressly stated otherwise.The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparing andadministering CPT-antibody immunoconjugates. Preferably, thecamptothecin is SN-38. The disclosed methods and compositions are of usefor the treatment of a variety of diseases and conditions which arerefractory or less responsive to other forms of therapy, and can includediseases against which suitable antibodies or antigen-binding antibodyfragments for selective targeting can be developed, or are available orknown, such as cancer.

Preferably, the targeting moiety is an antibody, antibody fragment,bispecific or other multivalent antibody, or other antibody-basedmolecule or compound. More preferably, the antibody or fragment is ananti-HLA-DR antibody or fragment. The antibody can be of variousisotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferablycomprising human IgG1 hinge and constant region sequences. Mostpreferably, the antibody is a human IgG4. The antibody or fragmentthereof can be a chimeric human-mouse, a chimeric human-primate, ahumanized (human framework and murine hypervariable (CDR) regions), orfully human antibody, as well as variations thereof, such as half-IgG4antibodies (referred to as “unibodies”), as described by van der NeutKolfschoten et al. (Science 2007; 317:1554-1557). More preferably, theantibody or fragment thereof may be designed or selected to comprisehuman constant region sequences that belong to specific allotypes, whichmay result in reduced immunogenicity when the immunoconjugate isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Where the disease state is cancer, many antigens expressed by orotherwise associated with tumor cells are known in the art, includingbut not limited to, carbonic anhydrase IX, alpha-fetoprotein (AFP),α-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733,BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1,CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67,CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138,CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12,HIF-1α, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met,DAM, DLL3, DLL4, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM,fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250antigen, GAGE, gp100, GRO-13, HLA-DR, HM1.24, human chorionicgonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia induciblefactor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12,IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, mesothelin,NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3,MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4antigen, pancreatic cancer mucin, PD-1 receptor, placental growthfactor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF,ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin,survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene marker andan oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CEACAM5, CEACAM6, EGP-1 (TROP-2), MUC-16, AFP, MUC5ac,CD74, CD19, CD20, CD22 or HLA-DR. The preferred anti-HLA-DR antibody maybe utilized alone, or in combination with another anti-TAA(tumor-associated antigen) antibody.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. Pat. No. 9,441,043), hPAM4 (anti-mucin, U.S.Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,151,164), hA19(anti-CD19, U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No.7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22,U.S. Pat. No. 5,789,554), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,772),hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S.Pat. No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 8,287,865),hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S.Pat. No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No.7,138,496), the Examples section of each cited patent or applicationincorporated herein by reference. More preferably, the antibody isIMMU-31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM5), hMN-3(anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). Most preferably, the antibody is hL243. As used herein, theterms epratuzumab and hLL2 are interchangeable, as are the termsveltuzumab and hA20 and the terms hL243g4P, hL243gamma4P and IMMU-114.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), pembrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), and Benlysta (HumanGenome Sciences).

In a preferred embodiment, the chemotherapeutic moiety is selected fromcamptothecin (CPT) and its analogs and derivatives and is morepreferably SN-38. However, other chemotherapeutic moieties that may beutilized include taxanes (e.g, baccatin III, taxol), epothilones,anthracyclines (e.g., doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolinodoxorubicin (2-PDOX) or a prodrug formof 2-PDOX (pro-2-PDOX); see, e.g., Priebe W (ed.), ACS symposium series574, published by American Chemical Society, Washington D.C., 1995 (332pp) and Nagy et al., Proc. Natl. Acad. Sci. USA 93:2464-2469, 1996),benzoquinoid ansamycins exemplified by geldanamycin (DeBoer et al.,Journal of Antibiotics 23:442-447, 1970; Neckers et al., Invest. NewDrugs 17:361-373, 1999), and the like. Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 chemotherapeutic moieties; more preferably 6 ormore chemotherapeutic moieties, more preferably 7 to 8, most preferablyabout 6 to about 12 chemotherapeutic moieties.

An example of a water soluble CPT derivative is CPT-11. Extensiveclinical data are available concerning CPT-11's pharmacology and its invivo conversion to the active SN-38 (Iyer and Ratain, Cancer ChemotherPharmacol. 42:S31-43 (1998); Mathijssen et al., Clin Cancer Res.7:2182-2194 (2002); Rivory, Ann NY Acad Sci. 922:205-215, 2000)). Theactive form SN-38 is about 2 to 3 orders of magnitude more potent thanCPT-11. In specific preferred embodiments, the immunoconjugate may be anhMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38, IMMU-31-SN-38, hRS7-SN-38,hA20-SN-38, hL243-SN-38, hLL1-SN-38 or hLL2-SN-38 conjugate.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphatic leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, T-celllymphomas and leukemias, multiple myeloma, Waldenstrom'smacroglobulinemia, carcinomas, melanomas, sarcomas, gliomas, bone, andskin cancers. The carcinomas may include carcinomas of the oral cavity,esophagus, gastrointestinal tract, pulmonary tract, lung, stomach,colon, breast, ovary, prostate, uterus, endometrium, cervix, urinarybladder, pancreas, bone, brain, connective tissue, liver, gall bladder,urinary bladder, kidney, skin, central nervous system and testes.

In certain embodiments involving treatment of cancer, the drugconjugates may be used in combination with surgery, radiation therapy,chemotherapy, immunotherapy with naked antibodies, radioimmunotherapy,immunomodulators, vaccines, and the like. These combination therapiescan allow lower doses of each therapeutic to be given in suchcombinations, thus reducing certain severe side effects, and potentiallyreducing the courses of therapy required. When there is no or minimaloverlapping toxicity, full doses of each can also be given.

Preferred optimal dosing of immunoconjugates may include a dosage ofbetween 3 mg/kg and 18 mg/kg, more preferably between 4 and 16 mg/kg,more preferably between 6 and 12 mg/kg, most preferably between 8 and 10mg/kg, preferably given either weekly, twice weekly or every other week.The optimal dosing schedule may include treatment cycles of twoconsecutive weeks of therapy followed by one, two, three or four weeksof rest, or alternating weeks of therapy and rest, or one week oftherapy followed by two, three or four weeks of rest, or three weeks oftherapy followed by one, two, three or four weeks of rest, or four weeksof therapy followed by one, two, three or four weeks of rest, or fiveweeks of therapy followed by one, two, three, four or five weeks ofrest, or administration once every two weeks, once every three weeks oronce a month. Treatment may be extended for any number of cycles,preferably at least 2, at least 4, at least 6, at least 8, at least 10,at least 12, at least 14, or at least 16 cycles. Exemplary dosages ofuse may include 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and 18 mg/kg. The person ofordinary skill will realize that a variety of factors, such as age,general health, specific organ function or weight, as well as effects ofprior therapy on specific organ systems (e.g., bone marrow) may beconsidered in selecting an optimal dosage of immunoconjugate, and thatthe dosage and/or frequency of administration may be increased ordecreased during the course of therapy. The dosage may be repeated asneeded, with evidence of tumor shrinkage observed after as few as 4 to 8doses. The optimized dosages and schedules of administration disclosedherein show unexpected superior efficacy and reduced toxicity in humansubjects, which could not have been predicted from animal model studies.Surprisingly, the superior efficacy allows treatment of tumors that werepreviously found to be resistant to one or more standard anti-cancertherapies, including the parental compound, CPT-11, from which SN-38 isderived in vivo.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugate,even with repeated infusions, with only relatively low-grade toxicitiesof nausea and vomiting observed, or manageable neutropenia. A furthersurprising result is the lack of accumulation of the antibody-drugconjugate, unlike other products that have conjugated SN-38 to albumin,PEG or other carriers. The lack of accumulation is associated withimproved tolerability and lack of serious toxicity even after repeatedor increased dosing. These surprising results allow optimization ofdosage and delivery schedule, with unexpectedly high efficacies and lowtoxicities. The claimed methods provide for shrinkage of solid tumors,in individuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by longest diameter). The person of ordinaryskill will realize that tumor size may be measured by a variety ofdifferent techniques, such as total tumor volume, maximal tumor size inany dimension or a combination of size measurements in severaldimensions. This may be with standard radiological procedures, such ascomputed tomography, ultrasonography, and/or positron-emissiontomography. The means of measuring size is less important than observinga trend of decreasing tumor size with immunoconjugate treatment,preferably resulting in elimination of the tumor.

While the immunoconjugate may be administered as a periodic bolusinjection, in alternative embodiments the immunoconjugate may beadministered by continuous infusion of antibody-drug conjugates. Inorder to increase the Cmax and extend the PK of the immunoconjugate inthe blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. More preferably,these immunoconjugates can be administered by intravenous infusions overrelatively short periods of 2 to 5 hours, more preferably 2-3 hours.

In particularly preferred embodiments, the immunoconjugates and dosingschedules may be efficacious in patients resistant to standardtherapies. For example, an hL243-SN-38 immunoconjugate may beadministered to a patient who has not responded to prior therapy withirinotecan, the parent agent of SN-38. Surprisingly, theirinotecan-resistant patient may show a partial or even a completeresponse to hL243-SN-38. The ability of the immunoconjugate tospecifically target the tumor tissue may overcome tumor resistance byimproved targeting and enhanced delivery of the therapeutic agent.Combinations of different SN-38 immunoconjugates, or SN-38-antibodyconjugates in combination with an antibody conjugated to a radionuclide,toxin or other drug, may provide even more improved efficacy and/orreduced toxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIG. 1. Structure of IMMU-140 (hL243-CL2A-SN-38).

FIG. 2. Comparative binding of IMMU-114 and IMMU-140. Binding curveswere obtained for the SN-38 conjugated (IMMU-140) and naked (IMMU-1140forms of hL243. A control non-specific antibody (h679) showed no bindingto HLA-DR+ cells.

FIG. 3. In vivo efficacy of IMMU-140 vs. IMMU-114 in MOLM-14 AMLxenografts.

FIG. 4. In vivo efficacy of IMMU-140 vs. IMMU-114 in MN-60 ALLxenografts.

FIG. 5. In vivo efficacy of IMMU-140 vs. IMMU-114 in CAG MM xenografts.

FIG. 6. In vivo efficacy of IMMU-140 vs. IMMU-114 in JVM-3 CLLxenografts.

FIG. 7. Binding of hL243-γ4P to human melanoma cells in vitro.

FIG. 8. Efficacy of IMMU-140 in human melanoma xenografts in vivo.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, immunoconjugates,drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases(including DNAses and RNAses), hormones, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radionuclides,oligonucleotides, interference RNA, siRNA, RNAi, anti-angiogenic agents,chemotherapeutic agents, cyokines, chemokines, prodrugs, enzymes,binding proteins or peptides or combinations thereof.

An immunoconjugate is an antibody, antigen-binding antibody fragment,antibody complex or antibody fusion protein that is conjugated to atherapeutic agent. Conjugation may be covalent or non-covalent.Preferably, conjugation is covalent.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

CPT is an abbreviation for camptothecin, and as used in the presentapplication CPT represents camptothecin itself or an analog orderivative of camptothecin, such as SN-38. The structures ofcamptothecin and some of its analogs, with the numbering indicated andthe rings labeled with letters A-E, are given in formula 1 in Chart 1below.

Camptothecin Conjugates

Non-limiting methods and compositions for preparing immunoconjugatescomprising a camptothecin therapeutic agent attached to an antibody orantigen-binding antibody fragment are described below. In preferredembodiments, the solubility of the drug is enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the antibody, wherein thedefined PEG is a low molecular weight PEG, preferably containing 1-30monomeric units, more preferably containing 1-12 monomeric units, mostpreferably 7-8 monomeric units.

Preferably, a first linker connects the drug at one end and mayterminate with an acetylene or an azide group at the other end. Thisfirst linker may comprise a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end. Said bifunctionaldefined PEG may be attached to the amine group of an amino alcohol, andthe hydroxyl group of the latter may be attached to the hydroxyl groupon the drug in the form of a carbonate. Alternatively, the non-azide (oracetylene) moiety of said defined bifunctional PEG is optionallyattached to the N-terminus of an L-amino acid or a polypeptide, with theC-terminus attached to the amino group of amino alcohol, and the hydroxygroup of the latter is attached to the hydroxyl group of the drug in theform of carbonate or carbamate, respectively.

A second linker, comprising an antibody-coupling group and a reactivegroup complementary to the azide (or acetylene) group of the firstlinker, namely acetylene (or azide), may react with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnisha final bifunctional drug product that is useful for conjugating todisease-targeting antibodies. The antibody-coupling group is preferablyeither a thiol or a thiol-reactive group.

Methods for selective regeneration of the 10-hydroxyl group in thepresence of the C-20 carbonate in preparations of drug-linker precursorinvolving CPT analogs such as SN-38 are provided below. Other protectinggroups for reactive hydroxyl groups in drugs such as the phenolichydroxyl in SN-38, for example t-butyldimethylsilyl ort-butyldiphenylsilyl, may also be used, and these are deprotected bytetrabutylammonium fluoride prior to linking of the derivatized drug toan antibody-coupling moiety. The 10-hydroxyl group of CPT analogs isalternatively protected as an ester or carbonate, other than ‘BOC’, suchthat the bifunctional CPT is conjugated to an antibody without priordeprotection of this protecting group. The protecting group is readilydeprotected under physiological pH conditions after the bioconjugate isadministered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’refers to a copper (+1)-catalyzed cycloaddition reaction between anacetylene moiety and an azide moiety (Kolb H C and Sharpless K B, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of clickchemistry are known and may be used. Click chemistry takes place inaqueous solution at near-neutral pH conditions, and is thus amenable fordrug conjugation. The advantage of click chemistry is that it ischemoselective, and complements other well-known conjugation chemistriessuch as the thiol-maleimide reaction.

While the present application focuses on use of antibodies or antibodyfragments as targeting moieties, the skilled artisan will realize thatwhere a conjugate comprises an antibody or antibody fragment, anothertype of targeting moiety, such as an aptamer, avimer, affibody orpeptide ligand, may be substituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,MAb-[L2]-[L1]-[AA]_(m)-[A′]-Drug  (2)where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4; and A′ is an additional spacer, selected from the groupof ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzyl alcohol, orsubstituted or unsubstituted ethylenediamine. The L amino acids of ‘AA’are selected from alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. If the A′ group contains hydroxyl, itis linked to the hydroxyl group or amino group of the drug in the formof a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, A′ is a substituted ethanolaminederived from an L-amino acid, wherein the carboxylic acid group of theamino acid is replaced by a hydroxymethyl moiety. A′ may be derived fromany one of the following L-amino acids: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine.

A preferred embodiment, referred to as MAb-CL2A-SN-38, is shown below.

Other embodiments are possible within the context of10-hydroxy-containing camptothecins, such as SN-38. In the example ofSN-38 as the drug, the more reactive 10-hydroxy group of the drug isderivatized leaving the 20-hydroxyl group unaffected. Within the generalformula 2, A′ is a substituted ethylenediamine.

In another preferred embodiment, the L1 component of the conjugatecontains a defined polyethyleneglycol (PEG) spacer with 1-30 repeatingmonomeric units. In a further preferred embodiment, PEG is a defined PEGwith 1-12 repeating monomeric units. The introduction of PEG may involveusing heterobifunctionalized PEG derivatives which are availablecommercially. The heterobifunctional PEG may contain an azide oracetylene group. An example of a heterobifunctional defined PEGcontaining 8 repeating monomeric units, with ‘NHS’ being succinimidyl,is given below in formula 3:

In a preferred embodiment, L2 has a plurality of acetylene (or azide)groups, ranging from 2-40, but preferably 2-20, and more preferably 2-5,and a single antibody-binding moiety.

A representative SN-38 conjugate of an antibody containing multiple drugmolecules and a single antibody-binding moiety is shown below. The ‘L2’component of this structure is appended to 2 acetylenic groups,resulting in the attachment of two azide-appended SN-38 molecules. Thebonding to MAb is represented as a succinimide.

In preferred embodiments, when the bifunctional drug contains athiol-reactive moiety as the antibody-binding group, the thiols on theantibody are generated on the lysine groups of the antibody using athiolating reagent. Methods for introducing thiol groups onto antibodiesby modifications of MAb's lysine groups are well known in the art (Wongin Chemistry of protein conjugation and cross-linking, CRC Press, Inc.,Boca Raton, Fla. (1991), pp 20-22). Alternatively, mild reduction ofinterchain disulfide bonds on the antibody (Willner et al., BioconjugateChem. 4:521-527 (1993)) using reducing agents such as dithiothreitol(DTT) can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region. In a morepreferred embodiment, attachment of SN-38 to reduced disulfidesulfhydryl groups results in formation of an antibody-SN-38immunoconjugate with 6 to 8 SN-38 moieties covalently attached perantibody molecule. Other methods of providing cysteine residues forattachment of drugs or other therapeutic agents are known, such as theuse of cysteine engineered antibodies (see U.S. Pat. No. 7,521,541, theExamples section of which is incorporated herein by reference.)

In alternative preferred embodiments, the chemotherapeutic moiety isselected from the group consisting of doxorubicin (DOX), epirubicin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyanomorpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX), Pro-2PDOX, CPT,10-hydroxy camptothecin, SN-38, topotecan, lurtotecan,9-aminocamptothecin, 9-nitrocamptothecin, taxanes, geldanamycin,ansamycins, and epothilones. In a more preferred embodiment, thechemotherapeutic moiety is SN-38. Preferably, in the conjugates of thepreferred embodiments, the antibody links to at least onechemotherapeutic moiety; preferably 1 to about 12 chemotherapeuticmoieties; more preferably about 6 to about 12 chemotherapeutic moieties,most preferably about 6 to 8 chemotherapeutic moieties.

Furthermore, in a preferred embodiment, the linker component ‘L2’comprises a thiol group that reacts with a thiol-reactive residueintroduced at one or more lysine side chain amino groups of saidantibody. In such cases, the antibody is pre-derivatized with athiol-reactive group such as a maleimide, vinylsulfone, bromoacetamide,or iodoacetamide by procedures well described in the art.

In the context of this work, a process was surprisingly discovered bywhich CPT drug-linkers can be prepared wherein CPT additionally has a10-hydroxyl group. This process involves, but is not limited to, theprotection of the 10-hydroxyl group as a t-butyloxycarbonyl (BOC)derivative, followed by the preparation of the penultimate intermediateof the drug-linker conjugate. Usually, removal of BOC group requirestreatment with strong acid such as trifluoroacetic acid (TFA). Underthese conditions, the CPT 20-O-linker carbonate, containing protectinggroups to be removed, is also susceptible to cleavage, thereby givingrise to unmodified CPT. In fact, the rationale for using a mildlyremovable methoxytrityl (MMT) protecting group for the lysine side chainof the linker molecule, as enunciated in the art, was precisely to avoidthis possibility (Walker et al., 2002). It was discovered that selectiveremoval of phenolic BOC protecting group is possible by carrying outreactions for short durations, optimally 3-to-5 minutes. Under theseconditions, the predominant product was that in which the ‘BOC’ at10-hydroxyl position was removed, while the carbonate at ‘20’ positionwas intact.

An alternative approach involves protecting the CPT analog's 10-hydroxyposition with a group other than ‘BOC’, such that the final product isready for conjugation to antibodies without a need for deprotecting the10-OH protecting group. The 10-hydroxy protecting group, which convertsthe 10-OH into a phenolic carbonate or a phenolic ester, is readilydeprotected by physiological pH conditions or by esterases after in vivoadministration of the conjugate. The faster removal of a phenoliccarbonate at the 10 position vs. a tertiary carbonate at the 20 positionof 10-hydroxycamptothecin under physiological condition has beendescribed by He et al. (He et al., Bioorganic & Medicinal Chemistry 12:4003-4008 (2004)). A 10-hydroxy protecting group on SN-38 can be ‘COR’where R can be a substituted alkyl such as “N(CH₃)₂—(CH₂)_(n)—” where nis 1-10 and wherein the terminal amino group is optionally in the formof a quaternary salt for enhanced aqueous solubility, or a simple alkylresidue such as “CH₃—(CH₂)_(n)—” where n is 0-10, or it can be an alkoxymoiety such as “CH₃—(CH₂)n-O—” where n is 0-10, or“N(CH₃)₂—(CH₂)_(n)—O—” where n is 2-10, or“R₁O—(CH₂—CH₂—O)_(n)—CH₂—CH₂—O—” where R₁ is ethyl or methyl and n is aninteger with values of 0-10. These 10-hydroxy derivatives are readilyprepared by treatment with the chloroformate of the chosen reagent, ifthe final derivative is to be a carbonate. Typically, the10-hydroxy-containing camptothecin such as SN-38 is treated with a molarequivalent of the chloroformate in dimethylformamide using triethylamineas the base. Under these conditions, the 20-OH position is unaffected.For forming 10-O-esters, the acid chloride of the chosen reagent isused.

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-X are as described in earlier sections, thebifunctional drug moiety, [L2]-[L1]-[AA]_(m)-[A-X]-Drug is firstprepared, followed by the conjugation of the bifunctional drug moiety tothe antibody (indicated herein as “MAb”).

In a preferred process of the preparation of a conjugate of a drugderivative and an antibody of the general formula 2, wherein thedescriptors L2, L1, AA and A-OH are as described in earlier sections,the bifunctional drug moiety is prepared by first linking A-OH to theC-terminus of AA via an amide bond, followed by coupling the amine endof AA to a carboxylic acid group of L1. If AA is absent (i.e. m=0), A-OHis directly attached to L1 via an amide bond. The cross-linker,[L1]-[AA]_(m)-[A-OH], is attached to drug's hydroxyl or amino group, andthis is followed by attachment to the L1 moiety, by taking recourse tothe reaction between azide (or acetylene) and acetylene (or azide)groups in L1 and L2 via click chemistry.

In one embodiment, the antibody is a monoclonal antibody (MAb). In otherembodiments, the antibody may be a multivalent and/or multispecific MAb.The antibody may be a murine, chimeric, humanized, or human monoclonalantibody, and said antibody may be in intact, fragment (Fab, Fab′,F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs) form, or ofan IgG1, IgG2a, IgG3, IgG4, IgA isotype, or submolecules therefrom.

In a preferred embodiment, the antibody binds to an antigen or epitopeof an antigen expressed on a cancer or malignant cell. The cancer cellis preferably a cell from a hematopoietic tumor, carcinoma, sarcoma,melanoma or a glial tumor. A preferred malignancy to be treatedaccording to the present invention is a malignant solid tumor orhematopoietic neoplasm.

In a preferred embodiment, the intracellularly-cleavable moiety may becleaved after it is internalized into the cell upon binding by theMAb-drug conjugate to a receptor thereof, and particularly cleaved byesterases and peptidases.

Anti-HLA-DR Antibodies

In preferred embodiments, the immunoconjugate comprises an anti-HLA-DRantibody, such as a humanized L243 antibody. Humanized L243 antibodiesbind to the same epitope on HLA-DR as the parental murine L243 antibody,but have reduced immunogenicity. mL243 is a monoclonal antibodypreviously described by Lampson & Levy (J Immunol, 1980, 125:293), whichhas been deposited at the American Type Culture Collection, Rockville,Md., under Accession number ATCC HB55.

The humanized L243 antibodies comprise the L243 heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO: 1), CDR2 (WINTYTREPTYADDFKG, SEQ IDNO:2) and CDR3 (DITAVVPTGFDY, SEQ ID NO:3) and the light chain CDRsequences CDR1 (RASENIYSNLA, SEQ ID NO:4), CDR2 (AASNLAD, SEQ ID NO:5),and CDR3 (QHFWTTPWA, SEQ ID NO:6), attached to human antibody FR andconstant region sequences. In more preferred embodiments, one or moremurine FR amino acid residues are substituted for the correspondinghuman FR residues, particularly at locations adjacent to or nearby theCDR residues. Exemplary murine V_(H) residues that may be substituted inthe humanized design are at positions: F27, K38, K46, A68 and F91.Exemplary murine V_(L) residues that may be substituted in the humanizeddesign are at positions R37, K39, V48, F49, and G1.

A particularly preferred form of hL243 antibody is illustrated in U.S.Pat. No. 7,612,180, incorporated herein by reference, which incorporatesFR sequences from the human RF-TS3, NEWM and REI antibodies. However, inother embodiments, the FR residues may be derived from any suitablehuman immunoglobulin, provided that the humanized antibody can fold suchthat it retains the ability to specifically bind HLA-DR. Preferably thetype of human framework (FR) used is of the same/similar class/type asthe donor antibody. More preferably, the human FR sequences are selectedto have a high degree of sequence homology with the corresponding murineFR sequences, particularly at positions spatially close or adjacent tothe CDRs. In accordance with this embodiment, the frameworks (ie, FR1-4)of the humanized L243 V_(H) or V_(L) may be derived from a combinationof human antibodies. Examples of human frameworks which may be used toconstruct CDR-grafted humanized antibodies are LAY, POM, TUR, TEI, KOL,NEWM, REI, RF and EU. Preferably human RF-TS3 FR1-3 and NEWM FR4 areused for the heavy chain and REI FR1-4 are used for the light chain. Thevariable domain residue numbering system used herein is described inKabat et al, (1991), Sequences of Proteins of Immunological Interest,5th Edition, United States Department of Health and Human Services

The light and heavy chain variable domains of the humanized antibodymolecule may be fused to human light or heavy chain constant domains.The human constant domains may be selected with regard to the proposedfunction of the antibody. In one embodiment, the human constant domainsmay be selected based on a lack of effector functions. The heavy chainconstant domains fused to the heavy chain variable region may be thoseof human IgA (α1 or α2 chain), IgG (γ1, γ2, γ3 or γ4 chain) or IgM (μchain). The light chain constant domains which may be fused to the lightchain variable region include human lambda and kappa chains.

In one particular embodiment of the present invention, a γ1 chain isused. In yet another particular embodiment, a γ4 chain is used. The useof the γ4 chain may in some cases increase the tolerance to hL243 insubjects (decreased side effects and infusion reactions, etc).

In one embodiment, analogues of human constant domains may be used.These include but are not limited to those constant domains containingone or more additional amino acids than the corresponding human domainor those constant domains wherein one or more existing amino acids ofthe corresponding human domain have been deleted or altered. Suchdomains may be obtained, for example, by oligonucleotide directedmutagenesis.

In a particular embodiment, an anti-HLA-DR antibody or fragment thereofmay be a fusion protein. The fusion protein may contain one or moreanti-HLA-DR antibodies or fragments thereof. In various embodiments, thefusion protein may also comprise one or more additional antibodiesagainst a different antigen, or may comprise a different effectorprotein or peptide, such as a cytokine. For example, the differentantigen may be a tumor marker selected from a B cell lineage antigen,(eg, CD19, CD20, or CD22) for the treatment of B cell malignancies. Inanother example, the different antigen may be expressed on other cellsthat cause other types of malignancies. Further, the cell marker may bea non-B cell lineage antigen, such as selected from the group consistingof HLA-DR, CD3, CD33, CD52, CD66, MUC1 and TAC.

In one embodiment, an anti-HLA-DR antibody may be combined with otherantibodies and used to treat a subject having or suspected of developinga disease. In accordance with this embodiment, an anti-HLA-DR antibodyor fragment thereof may be combined with an anticancer monoclonalantibody such as a humanized monoclonal antibody (eg hA20, anti-CD20Mab) and used to treat cancer. It is contemplated herein that ananti-HLA-DR antibody may be used as a separate antibody composition incombination with one or more other separate antibody compositions, orused as a bi-functional antibody containing, for example, oneanti-HLA-DR and one other anti-tumor antibody, such as hA20. In anotherparticular embodiment, the antibody may target a B cell malignancy. TheB cell malignancy may consist of indolent forms of B cell lymphomas,aggressive forms of B cell lymphomas, chronic lymphatic leukemias, acutelymphatic leukemias, Waldenstrom's macroglobulinemia, and multiplemyeloma. Other non-malignant B cell disorders and related diseases thatmay be treated with the subject antibodies include many autoimmune andimmune dysregulatory diseases, such as septicemia and septic shock.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et a. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising a human antigen, such as human HLA-DR,removing the spleen to obtain B-lymphocytes, fusing the B-lymphocyteswith myeloma cells to produce hybridomas, cloning the hybridomas,selecting positive clones which produce antibodies to the human antigen,culturing the clones that produce antibodies to the antigen, andisolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents, such as camptothecins, using the techniquesdisclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XENOMOUSE® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5 S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. No. 4,036,945; U.S. Pat. No. 4,331,647; Nisonoff et al., 1960,Arch. Biochem. Biophys., 89:230; Porter, 1959, Biochem. J., 73:119;Edelman et al., 1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press),and Coligan et al. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (JohnWiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Known Antibodies

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. Pat. No. 9,441,043), hPAM4 (anti-mucin, U.S.Pat. No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,151,164), hA19(anti-CD19, U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No.7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22,U.S. Pat. No. 5,789,554), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,772),hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S.Pat. No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 8,287,865),hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S.Pat. No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No.7,138,496), the Examples section of each cited patent or applicationincorporated herein by reference. More preferably, the antibody isIMMU-31 (anti-AFP), hRS7 (anti-TROP-2), hMN-14 (anti-CEACAM5), hMN-3(anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). As used herein, the terms epratuzumab and hLL2 areinterchangeable, as are the terms veltuzumab and hA20, and the termshL243g4P, hL243gamma4P and IMMU-114.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), pembrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), natalizumab (anti-α4integrin), omalizumab (anti-IgE); anti-TNF-α antibodies such as CDP571(Ofei et al., 2011, Diabetes 45:881-85), MTNFAI, M2TNFAI, M3TNFAI,M3TNFABI, M302B, M303 (Thermo Scientific, Rockford, Ill.), infliximab(Centocor, Malvern, Pa.), certolizumab pegol (UCB, Brussels, Belgium),anti-CD40L (UCB, Brussels, Belgium), adalimumab (Abbott, Abbott Park,Ill.), or Benlysta (Human Genome Sciences).

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Penis, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Studies with checkpoint inhibitor antibodies for cancer therapy havegenerated unprecedented response rates in cancers previously thought tobe resistant to cancer treatment (see, e.g., Ott & Bhardwaj, 2013,Frontiers in Immunology 4:346; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85; Pardoll, 2012, Nature Reviews 12:252-264; Mavilio & Lugli,).Therapy with antagonistic checkpoint blocking antibodies against CTLA-4,PD-1 and PD-L1 are one of the most promising new avenues ofimmunotherapy for cancer and other diseases. In contrast to the majorityof anti-cancer agents, checkpoint inhibitor do not target tumor cellsdirectly, but rather target lymphocyte receptors or their ligands inorder to enhance the endogenous antitumor activity of the immune system(Pardoll, 2012, Nature Reviews 12:252-264). Because such antibodies actprimarily by regulating the immune response to diseased cells, they maybe used in combination with other therapeutic modalities, such as thesubject anti-HLA-DR antibodies, to enhance their anti-tumor effect.

Programmed cell death protein 1 (PD-1, also known as CD279) encodes acell surface membrane protein of the immunoglobulin superfamily, whichis expressed in B cells and NK cells (Shinohara et al., 1995, Genomics23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45;Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature Reviews12:252-264). Anti-PD1 antibodies have been used for treatment ofmelanoma, non-small-cell lung cancer, bladder cancer, prostate cancer,colorectal cancer, head and neck cancer, triple-negative breast cancer,leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N EnglJ Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Bergeret al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013,Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85).

Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, MERCK),nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), and pidilizumab (CT-011,CURETECH LTD.). Anti-PD1 antibodies are commercially available, forexample from ABCAM® (AB137132), BIOLEGEND® (EH12.2H7, RMPI-14) andAFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).

Programmed cell death 1 ligand 1 (PD-L1, also known as CD274) is aligand for PD-1, found on activated T cells, B cells, myeloid cells andmacrophages. The complex of PD-1 and PD-L1 inhibits proliferation ofCD8+ T cells and reduces the immune response (Topalian et al., 2012, NEngl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65).Anti-PDL1 antibodies have been used for treatment of non-small cell lungcancer, melanoma, colorectal cancer, renal-cell cancer, pancreaticcancer, gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies (Brahmer et al., N Eng J Med 366:2455-65; Ott et al., 2013,Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger etal., 2008, Clin Cancer Res 14:13044-51).

Exemplary anti-PDL1 antibodies include MDX-1105 (MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).Anti-PDL1 antibodies are also commercially available, for example fromAFFYMETRIX EBIOSCIENCE (MIH1).

Cytotoxic T-lymphocyte antigen 4 (CTLA-4, also known as CD152) is also amember of the immunoglobulin superfamily that is expressed exclusivelyon T-cells. CTLA-4 acts to inhibit T cell activation and is reported toinhibit helper T cell activity and enhance regulatory T cellimmunosuppressive activity (Pardoll, 2012, Nature Reviews 12:252-264).Anti-CTL4A antibodies have been used in clinical trials for treatment ofmelanoma, prostate cancer, small cell lung cancer, non-small cell lungcancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al.,2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wadaet al., 2013, J Transl Med 11:89).

Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-MyersSquibb) and tremelimumab (PFIZER). Anti-PD1 antibodies are commerciallyavailable, for example from ABCAM® (AB134090), SINO BIOLOGICAL INC.(11159-H03H, 11159-H08H), and THERMO SCIENTIFIC PIERCE (PA5-29572,PA5-23967, PA5-26465, MA1-12205, MA1-35914). Ipilimumab has recentlyreceived FDA approval for treatment of metastatic melanoma (Wada et al.,2013, J Transl Med 11:89).

These and other known checkpoint inhibitor antibodies may be used incombination with anti-HLA-DR antibodies alone or in further combinationwith an interferon, such as interferon-α, for improved cancer therapy.

The person of ordinary skill will be aware that it is possible togenerate any number of antibodies against a known and well characterizedtarget antigen, such as human HLA-DR. The human HLA-DR antigen has beenwell characterized in the art, for example by its amino acid sequence(see, e.g., GenBank Accession No. ADM15723.1).

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3× anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3× anti-HLA-DR. In certainembodiments, the techniques and compositions for therapeutic agentconjugation disclosed herein may be used with bispecific ormultispecific antibodies as the targeting moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of each ofwhich is incorporated herein by reference. Reduction of the peptidelinker length to less than 12 amino acid residues prevents pairing ofV_(H) and V_(L) domains on the same chain and forces pairing of V_(H)and V_(L) domains with complementary domains on other chains, resultingin the formation of functional multimers. Polypeptide chains of V_(H)and V_(L) domains that are joined with linkers between 3 and 12 aminoacid residues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabody) andtetramers (termed tetrabody) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as DOCK-AND-LOCK® (DNL®) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens.

Such antibodies need not only be used in combination, but can becombined as fusion proteins of various forms, such as IgG, Fab, scFv,and the like, as described in U.S. Pat. Nos. 6,083,477; 6,183,744 and6,962,702 and U.S. Patent Application Publication Nos. 20030124058;20030219433; 20040001825; 20040202666; 20040219156; 20040219203;20040235065; 20050002945; 20050014207; 20050025709; 20050079184;20050169926; 20050175582; 20050249738; 20060014245 and 20060034759, theExamples section of each incorporated herein by reference.

DOCK AND LOCK® (DNL®)

In certain embodiments, the anti-HLA-DR antibodies or fragments may beincorporated into a multimeric complex, for example using a techniquereferred to as DOCK-AND-LOCK® (DNL®). The method exploits specificprotein/protein interactions that occur between the regulatory (R)subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain(AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al., FEBSLetters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathways triggered by the binding of thesecond messenger cAMP to the R subunits, was first isolated from rabbitskeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763).The structure of the holoenzyme consists of two catalytic subunits heldin an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RIand RII), and each type has α and β isoforms (Scott, Pharmacol. Ther.1991; 50:123). The R subunits have been isolated only as stable dimersand the dimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561).

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various suB cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188), with binding affinities reported for RII dimersranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445). Interestingly, AKAPs will only bind to dimeric R subunits.For human RIIc, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

DDD of Human RIIα and AD of AKAPs as Linker Modules

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAP proteins as an excellent pair of linker modules fordocking any two entities, referred to hereafter as A and B, into anoncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chimura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically.

In preferred embodiments, the anti-HLA-DR MAb DNL constructs may bebased on a variation of the a₂b structure, in which an IgGimmunoglobulin molecule (e.g., hL243) is attached at its C-terminal endto two copies of an AD moiety. Preferably the AD moiety is attached tothe C-terminal end of each light chain. Each AD moiety is capable ofbinding to two DDD moieties in the form of a dimer. By attaching acytokine or other therapeutic protein or peptide to each DDD moiety,four copies of cytokine or other therapeutic moiety are conjugated toeach IgG molecule.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances. The DNL method was disclosed inU.S. Pat. Nos. 7,550,143; 7,521,056; 76,534,866; 7,527,787 and7,666,400, the Examples section of each incorporated herein byreference.

In preferred embodiments, the effector moiety is a protein or peptide,more preferably an antibody, antibody fragment or cytokine, which can belinked to a DDD or AD unit to form a fusion protein or peptide. Avariety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

DDD and AD Sequence Variants

In certain embodiments, the AD and DDD sequences incorporated into theanti-HLA-DR MAb DNL complex comprise the amino acid sequences of DDD1(SEQ ID NO:7) and AD1 (SEQ ID NO:9) below. In more preferredembodiments, the AD and DDD sequences comprise the amino acid sequencesof DDD2 (SEQ ID NO:8) and AD2 (SEQ ID NO:10), which are designed topromote disulfide bond formation between the DDD and AD moieties.

DDD1 (SEQ ID NO: 7) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 8) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 9) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 10)CGQIEYLAKQIVDNAIQQAGC

However, in alternative embodiments sequence variants AD and/or DDDmoieties may be utilized in construction of the anti-HLA-DR MAb DNLcomplexes. The structure-function relationships of the AD and DDDdomains have been the subject of investigation. (See, e.g., Burns-Hamuroet al., 2005, Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50;Hundsrucker et al., 2006, Biochem J 396:297-306; Stokka et al., 2006,Biochem J 400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kindermanet al., 2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined below in SEQ IDNO:7. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding. Conservative amino acid substitutions are discussed inmore detail below, but could involve for example substitution of anaspartate residue for a glutamate residue, or a leucine or valineresidue for an isoleucine residue, etc. Such conservative amino acidsubstitutions are well known in the art.

Human DDD sequence from protein kinase A (SEQ ID NO: 7)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:9), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:9.

AKAP-IS sequence (SEQ ID NO: 9) QIEYLAKQIVDNAIQQA

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO: 11), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare anti-HLA-DR MAb DNL constructs. It is anticipatedthat, as with the AKAP-IS sequence shown in SEQ ID NO:9, the AD moietymay also include the additional N-terminal residues cysteine and glycineand C-terminal residues glycine and cysteine, as shown in SEQ ID NO: 10.

SuperAKAP-IS (SEQ ID NO: 11) QIEYVAKQIVDYAIHQA

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:9). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.)

AKAP-IS (SEQ ID NO: 9) QIEYLAKQIVDNAIQQA

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:7. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins.

(SEQ ID NO: 7) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids D12 (Kabat position356) and L14 (Kabat position 358) in the CH3 sequence of the heavy chainIgG1. The nG1m1 allotype comprises the amino acids E12 and M14 at thesame locations. Both G1m1 and nG1m1 allotypes comprise an E13 residue inbetween the two variable sites and the allotypes are sometimes referredto as DEL and EEM allotypes. A non-limiting example of the heavy chainconstant region sequences for G1m1 and nG1m1 allotype antibodies isshown for the exemplary antibodies rituximab (SEQ ID NO: 12) andveltuzumab (SEQ ID NO: 13).

Rituximab heavy chain variable region sequence (SEQ ID NO. 12)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KVeltuzumab heavy chain variable region (SEQ ID NO: 13)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the EEM allotype, with a glutamate residueat Kabat position 356, a methionine at Kabat position 358, andpreferably an arginine residue at Kabat position 214. Surprisingly, itwas found that repeated subcutaneous administration of G1m3 antibodiesover a long period of time did not result in a significant immuneresponse.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL® constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within +2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp (See, e.g., PROWL website at rockefeller.edu). For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Conjugation Protocols

In certain embodiments, the anti-HLA-DR antibody or fragment may beconjugated to one or more therapeutic or diagnostic agents. Thetherapeutic agents do not need to be the same but can be different, e.g.a drug and a radioisotope. For example, ¹³¹I can be incorporated into atyrosine of an antibody or fusion protein and a drug attached to anepsilon amino group of a lysine residue. Therapeutic and diagnosticagents also can be attached, for example to reduced SH groups and/or tocarbohydrate side chains. Many methods for making covalent ornon-covalent conjugates of therapeutic or diagnostic agents withantibodies or fusion proteins are known in the art and any such knownmethod may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.patent application Ser. No. 12/112,289, incorporated herein by referencein its entirety).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,⁹⁹mTc, ⁹⁴mTc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The same chelates,when complexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA,and TETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates such as macrocyclic polyethers, which are of interest forstably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F—Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. patentapplication Ser. No. 12/112,289, filed Apr. 30, 2008, the entire text ofwhich is incorporated herein by reference.

In preferred embodiments, the conjugation protocol is based on athiol-maleimide, a thiol-vinylsulfone, a thiol-bromoacetamide, or athiol-iodoacetamide reaction that is facile at neutral or acidic pH.This obviates the need for higher pH conditions for conjugations as, forinstance, would be necessitated when using active esters. Furtherdetails of exemplary conjugation protocols are described below in theExamples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofa therapeutic conjugate as described herein to a subject. Diseases thatmay be treated with the therapeutic conjugates described herein include,but are not limited to B-cell malignancies (e.g., non-Hodgkin'slymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphatic leukemia, chronic lymphatic leukemia, hairy cellleukemia) using an anti-HLA-DR immunoconjugate. More preferably, thecancer is AML (acute myelocytic leukemia), ALL (acute lymphocyticleukemia) or MM (multiple myeloma). However, any HLA-DR positive tumormay be treated with the subject immunoconjugates, such as skin,esophageal, stomach, colon, rectal, pancreatic, lung, breast, ovarian,bladder, endometrial, cervical, testicular, melanoma, kidney, or livercancer. Such therapeutics can be given once or repeatedly, depending onthe disease state and tolerability of the conjugate, and can also beused optionally in combination with other therapeutic modalities, suchas surgery, external radiation, radioimmunotherapy, immunotherapy,chemotherapy, antisense therapy, interference RNA therapy, gene therapy,and the like. Each combination will be adapted to the tumor type, stage,patient condition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In an exemplary embodiment, an hL243 antibody is a humanized antibodycomprising the heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO: 1),CDR2 (WINTYTREPTYADDFKG, SEQ ID NO:2), and CDR3 (DITAVVPTGFDY, SEQ IDNO:3) and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ ID NO:4),CDR2 (AASNLAD, SEQ ID NO:5), and CDR3 (QHFWTTPWA, SEQ ID NO:6).

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

In another preferred embodiment, the therapeutic conjugates can be usedto treat autoimmune disease or immune system dysfunction (e.g.,graft-versus-host disease, organ transplant rejection). Antibodies ofuse to treat autoimmune/immune dysfunction disease may bind to exemplaryantigens including, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2,CD3, CD4, CD5, CD7, CD8, CD10, CD11 b, CD11c, CD13, CD14, CD15, CD16,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L,CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR. Antibodies that bind to these andother target antigens, discussed above, may be used to treat autoimmuneor immune dysfunction diseases. Autoimmune diseases that may be treatedwith immunoconjugates may include acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In another preferred embodiment, a therapeutic agent used in combinationwith the camptothecin conjugate of this invention may comprise one ormore isotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins,taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatinum,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide,transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839. Such agents may be part of theconjugates described herein or may alternatively be administered incombination with the described conjugates, either prior to,simultaneously with or after the conjugate. Alternatively, one or moretherapeutic naked antibodies as are known in the art may be used incombination with the described conjugates. Exemplary therapeutic nakedantibodies are described above.

In preferred embodiments, a therapeutic agent to be used in combinationwith a DNA-breaking antibody conjugate (e.g., an SN-38-ADC) is amicrotubule inhibitor, such as a vinca alkaloid, a taxanes, amaytansinoid or an auristatin. Exemplary known microtubule inhibitorsinclude paclitaxel, vincristine, vinblastine, mertansine, epothilone,docetaxel, discodermolide, combrestatin, podophyllotoxin, CI-980,phenylahistins, steganacins, curacins, 2-methoxy estradiol, E7010,methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine,dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins,hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate.

In an alternative preferred embodiment, a therapeutic agent to be usedin combination with a DNA-breaking ADC, such as an SN-38-antibodyconjugate, is a PARP inhibitor, such as olaparib, talazoparib (BMN-673),rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699,BSI-201, CEP-8983 or 3-aminobenzamide.

In another alternative, a therapeutic agent used in combination with anantibody or immunoconjugate is a Bruton kinase inhibitor, such as suchas ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059,GDC-0834, LFM-A13 or RN486.

In yet another alternative, a therapeutic agent used in combination withan antibody or immunoconjugate is a PI3K inhibitor, such as idelalisib,Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib),BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120,XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114,TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), ranpirnase, DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. (See, e.g., Pastan. et al., Cell(1986), 47:641, and Sharkey and Goldenberg, C A Cancer J Clin. 2006July-August; 56(4):226-43.) Additional toxins suitable for use hereinare known to those of skill in the art and are disclosed in U.S. Pat.No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, an hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β, -γ or -λ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

The person of ordinary skill will realize that the subjectimmunoconjugates, comprising a camptothecin conjugated to an antibody orantibody fragment, may be used alone or in combination with one or moreother therapeutic agents, such as a second antibody, second antibodyfragment, second immunoconjugate, radionuclide, toxin, drug,chemotherapeutic agent, radiation therapy, chemokine, cytokine,immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA. Suchadditional therapeutic agents may be administered separately, incombination with, or attached to the subject antibody-drugimmunoconjugates.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, subcutaneous, rectal, transmucosal,intestinal administration, intramuscular, intramedullary, intrathecal,direct intraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

In a preferred embodiment, the immunoconjugate is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate from such a matrix depends upon the molecular weightof the immunoconjugate, the amount of immunoconjugate within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered immunoconjugate for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. A dosage of1-20 mg/kg for a 70 kg patient, for example, is 70-1,400 mg, or 41-824mg/m² for a 1.7-m patient. The dosage may be repeated as needed, forexample, once per week for 4-10 weeks, once per week for 8 weeks, oronce per week for 4 weeks. It may also be given less frequently, such asevery other week for several months, or monthly or quarterly for manymonths, as needed in a maintenance therapy. Preferred dosages mayinclude, but are not limited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, and 18 mg/kg. Thedosage is preferably administered multiple times, once or twice a week.A minimum dosage schedule of 4 weeks, more preferably 8 weeks, morepreferably 16 weeks or longer may be used. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an immunoconjugate may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient), it may beadministered once or even twice weekly for 4 to 10 weeks. Alternatively,the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3months. It has been determined, however, that even higher doses, such as12 mg/kg once weekly or once every 2-3 weeks can be administered by slowi.v. infusion, for repeated dosing cycles. The dosing schedule canoptionally be repeated at other intervals and dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

In certain preferred embodiments, the SN-38 conjugated anti-HLA-DR maybe administered subcutaneously. For subcutaneous administration, dosagesof ADCs such as IMMU-140 (hL243-CL2A-SN-38) may be limited by theability to concentrate the ADC without precipitation or aggregation, aswell as the volume of administration that may be given subcutaneously(preferably, 1, 2, or 3 ml or less). Consequently, for subcutaneousadministration the ADC may be given at 2 to 4 mg/kg, given daily for 1week, or 3 times weekly for 2 weeks, or twice weekly for two weeks,followed by rest. Maintenance doses of ADC may be administered i.v. ors.c. every two to three weeks or monthly after induction. Alternatively,induction may occur with two to four cycles of i.v. administration at8-10 mg/kg (each cycle with ADC administration on Days 1 and 8 of two21-day cycles with a one-week rest period in between), followed by s.c.administration as active dosing one or more times weekly or asmaintenance therapy. Dosing may be adjusted based on interim tumor scansand/or by analysis of Trop-2 positive circulating tumor cells.

In preferred embodiments, the immunoconjugates are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with immunoconjugates mayinclude acute and chronic immune thrombocytopenias, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one conjugated antibody or other targeting moiety as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

General

Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; MMT, monomethoxytrityl;PABOH, p-aminobenzyl alcohol; PEG, polyethylene glycol; SMCC,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TBAF,tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl chloride.

Chloroformates of hydroxy compounds in the following examples wereprepared using triphosgene and DMAP according to the procedure describedin Moon et al. (J. Medicinal Chem. 51:6916-6926, 2008). Extractivework-up refers to extraction with chloroform, dichloromethane or ethylacetate, and washing optionally with saturated bicarbonate, water, andwith saturated sodium chloride. Flash chromatography was done using230-400 mesh silica gel and methanol-dichloromethane gradient, using upto 15% v/v methanol-dichloromethane, unless otherwise stated. Reversephase HPLC was performed by Method A using a 7.8×300 mm C18 HPLC column,fitted with a precolumn filter, and using a solvent gradient of 100%solvent A to 100% solvent B in 10 minutes at a flow rate of 3 mL perminute and maintaining at 100% solvent B at a flow rate of 4.5 mL perminute for 5 or 10 minutes; or by Method B using a 4.6×30 mm XbridgeC18, 2.5 m, column, fitted with a precolumn filter, using the solventgradient of 100% solvent A to 100% of solvent B at a flow rate of 1.5 mLper minutes for 4 min and 100% of solvent B at a flow rate of 2 mL perminutes for 1 minutes. Solvent A was 0.3% aqueous ammonium acetate, pH4.46 while solvent B was 9:1 acetonitrile-aqueous ammonium acetate(0.3%), pH 4.46. HPLC was monitored by a dual in-line absorbancedetector set at 360 nm and 254 nm.

Example 1. Efficacy of Anti-HLA-DR Antibody Drug Conjugate IMMU-140(hL243-CL2A-SN-38) in HLA-DR⁺ Cancers In Vitro and In Vivo

Relapsed AML (acute myelocytic leukemia), ALL (acute lymphocyticleukemia) and MM (multiple myeloma) continue to be a therapy challenge.IMMU-114 (hL243) is a humanized anti-HLA-DR IgG₄ monoclonal antibodyengineered to lack effector-cell functions, but retains HLA-DR bindingand a broad range of antitumor effects in diverse hematologicalneoplasms (Stein R et al., Blood. 2010; 115:5180-90). When givensubcutaneously, it demonstrated efficacy in an initial Phase I clinicaltrial in relapsed or refractory NHL and CLL, with a good safety profile(ClinicalTrials.gov, NCT01728207).

In vitro, AML has proven to be resistant to the antitumor effects ofIMMU-114, despite high expression levels of HLA-DR. Likewise, in severaldifferent human ALL, CLL, and MM cell lines, IMMU-114 demonstrated arange of antitumor effects from a low of 9% to a high of 69%.

In an effort to improve the antitumor activity of IMMU-114, anantibody-drug conjugate (ADC), termed IMMU-140, was made in whichIMMU-114 was conjugated with the active metabolite of irinotecan, SN-38.Another ADC utilizing SN-38 (sacituzumab govitecan) being studied insolid tumors has been well tolerated, with clinically significantobjective responses in patients given multiple cycles over >6 months,with manageable neutropenia being the major toxicity. Thus, our goal wasto determine if SN-38, a drug not commonly used in hematopoieticcancers, would prove to be an effective and safe therapeutic whentargeted with the IMMU-114 antibody.

In this current study, the in vitro and in vivo activity of hL243-SN-38(IMMU-140) versus parental IMMU-114 was examined in human AML, ALL, MM,and CLL xenografts.

Methods

The method used for the conjugation of SN-38 to hL243 IgG₄ has beenpreviously described and resulted in a drug-to-antibody-ratio range of6.1 to 6.6 (Moon S J et al. J. Med. Chem. 2008; 51:6916-26). The SN-38linker (below, on left) contains a short polyethylene glycol (PEG)moiety to confer aqueous solubility. A maleimide group was incorporatedfor fast thiol-maleimide conjugation to mildly reduced antibody. Abenzylcarbonate site provided a pH-mediated cleavage site to release thedrug from the linker. The cross-linker was attached to SN-38's20-hydroxy position, to keep the lactone ring of the drug from openingto the less active carboxylic acid form under physiological conditions.The structure of the ADC is shown in FIG. 1.

The conjugate was characterized by size-exclusion HPLC (not shown).Unmodified IMMU-114 and IMMU-140 conjugate with a drug/antibody molarsubstitution of 6.1 were compared (not shown). Unmodified IMMU-114 wasdetected at 280 nm, and the conjugate was detected at the absorbancewavelength of SN-38, namely 360 nm. The conjugate was >98% monomeric(not shown).

There was no evidence of any loss of binding specificity of the ADC, asdemonstrated by comparable binding of IMMU-140 and IMMU-114 to an HLA-DRpositive human melanoma cell line (A-374) via a cell-based ELISA (FIG.2). Both recognize the a-chain when it associates with the b-chain.K_(D)-values are shown in the Table 2 below. Antibody control (h679) isa humanized anti-histamine-succinyl-glycine (HSG) IgG.

TABLE 2 Binding affinities of IMMU-140 vs. IMMU-114. K_(D) (nM) 95% C.I.R² IMMU-140 0.77 0.62 to 0.91 0.97 IMMU-114 0.65 0.53 to 0.77 0.97

For in vitro cytotoxicity assays, cells were plated in 96-well plates(1×10⁴ cells/well) followed by addition of each test agent: Free SN-38(2.5×10⁻⁷ to 3.8×10⁻¹² M), IMMU-140 (2.5×10⁻⁷ to 3.8×10⁻¹² M, SN-38equivalents), and IMMU-114 (4×10⁻⁸ to 6.2×10⁻¹³ M). Plates wereincubated for 96 h before cell viability determined by MTS. Inhibitiondetermined as percent viable cells in treated wells compared tountreated controls.

Apoptosis signaling was determined in cells (2×10⁶) treated with 10 nMprotein concentration of either IMMU-114 or IMMU-140 before being lysedand proteins (25 mg) resolved via SDS-PAGE. Proteins were transferred toPVD membranes for Western blotting.

For AML and MM disease models, NSG/SCID and C.B.-17 SCID mice received 2Gy irradiation 24 h prior to an i.v. injection of MOLM-14 (2×10⁶) or CAGcells (1×10⁷), respectively.

ALL (MN-60) and CLL (JVM-3) were established in C.B-17 SCID miceinjected i.v. with 1×10⁷ cells.

All therapies began 5 days post-tumor-cell injection. Test agents,including non-targeting control SN-38-ADCs, were administered at dosesindicated in the figures (100 to 500 mg), twice-weekly for 4 wks.Animals were sacrificed at disease progression, characterized by theonset of hind-limb paralysis or loss of more than 15% body weight.

Results

HLA-DR expression was examined in various human cancer cell lines. HumanALL, MM, CLL, and AML cell lines were harvested from tissue culture andanalyzed by FACS for HLA-DR (alpha chain) expression usingAlexaFluor-647-labeled IMMU-114. Mean fluorescence intensity (MFI) ofIMMU-114 and non-targeting control h679 antibody demonstrated highexpression of HLA-DR in all four cell lines, with MFI values forIMMU-114 of 6.58×10⁴ (MN-60 ALL cell line), 8.07×10⁴ (CAG MM cell line),7.87×10⁴ (JVM-3 CLL cell line) and 5.75×10³ (MOLM-14 AML cell line). Bycomparison, CAG, MN-60, and JVM-3 exhibit >10-fold higher expressioncompared to the AML cell line, MOLM-14.

In vitro, IMMU-140 achieved IC₅₀-values at low nM concentrations(ranging from 0.8 to 7.1 nM) in all four hematopoietic tumor types (ALL,MM, CLL, and AML), as shown in Table 3 below. In vitro, JVM-3demonstrated the most sensitivity to both IMMU-140 and IMMU-114. Theremaining three cell lines only exhibited 50% or greatergrowth-inhibition in the presence of SN-38 and IMMU-140. While 50%inhibition was not achieved in those cell lines with IMMU-114, theunconjugated antibody mediated significant growth inhibition in both CAG(MM) and MN-60 (ALL) at 40 nM when compared to a non-targeting controlantibody. As was reported previously with two other AML cell lines(Stein R et al., Blood. 2010; 115:5180-90), MOLM-14 also was resistantto IMMU-114, but was sensitive to IMMU-140.

TABLE 3 In vitro cytotoxicity of IMMU-140 vs. IMMU-114 in AML, ALL, CLLand MM cells. IMMU-114 Maximum Free SN-38 IMMU-140^(‡) IC₅₀ % InhibitionCell Line Disease IC₅₀ (nM) IC₅₀ (nM) (nM) at 40 nM JVM-3 CLL 0.51 ±0.18 0.77 ± 0.15 1.52 ± 0.84 ^(†)65 ± 11 CAG MM 7.02 ± 1.77 7.05 ±2.73 >40 ^(†)28 ± 6  MN-60 ALL 0.86 ± 0.09 1.29 ± 0.27 >40 ^(†)37 ± 10MOLM-14 AML *0.90 ± 0.13  1.21 ± 0.08 >40 11 ± 5  ^(‡)Concentration ofIMMU-140 shown as SN-38 equivalents *IC₅₀ of free SN-38 is significantlydifferent compared to IMMU-140 in MOLM-14 (P = 0.0266) ^(†)Significantinhibition compared to control antibody (P < 0.0061)

IMMU-140 demonstrated dual-apoptosis signaling pathways mediated throughits anti-HLA-DR binding of target cells and delivery of SN-38. IMMU-114signals apoptosis via p-ERK-1/2 and apoptosis-inducing factor (AIF) inNHL, ALL, MM, and CLL, but not AML (Stein R et al. Blood. 2010;115(25):5180-5190). Here we demonstrated that both IMMU-114 and IMMU-140are capable of mediating the phosphorylation of ERK1/2 and up-regulatingAIF in three different hematopoietic cell lines (not shown), includingthe AML cell line MOLM-14 (not shown), suggesting defects in othersignaling components of this pathway in AML since it is insensitive toIMMU-114 both in vitro and in vivo.

Importantly, IMMU-140, through its SN-38 payload, also mediated PARPcleavage in all three cell lines (not shown), including MOLM-14. Theresulting double-stranded DNA (dsDNA) breaks, as evidenced by increasedp-H2A.X levels, were most evident in the cells treated with IMMU-140(not shown).

In experimental MOLM-14 AML, saline control and IMMU-114 treated micesuccumbed to disease progression quickly, with a median survival time(MST) of only 14 and 15 days, respectively (FIG. 3). Conversely, micetreated with IMMU-140 had a greater than 1.5-fold increase in survival(MST=37 d, P=0.0031) (FIG. 3). Further, a dose-reduction to 250 mgIMMU-140 still provided a statistically significant, greater than 80%improvement in survival compared to saline and control ADC(anti-CEA-SN-38 IMMU-130) administered at the same dose (MST=21 d,P=0.0031) (FIG. 3).

In mice bearing MN-60 ALL xenografts (FIG. 4), IMMU-114 provided a >60%improvement in survival compared to saline control (MST=37 d vs. 22.5 d,respectively; P<0.0001), whereas IMMU-140 increased this by another 80%(MST=66.5 d) which was significantly better than all other treatments,including IMMU-114 (P<0.0001) (FIG. 4). Therapy with IMMU-140 was welltolerated by the mice with no appreciable loss in body weight.

Mice with CAG MM xenografts (FIG. 5) had a greater than 151-d MST whentreated with IMMU-140 compared to 32 d for saline control (P<0.0001).This survival benefit also was significantly higher than for micetreated with bortezomib (0.89 mg/kg) or control ADC+ bortezomib(MST=32.5 d for both; P<0.0001) (FIG. 5). While not significant,IMMU-140 does provide a >60% improvement in survival when compared toIMMU-114 therapy (MST=94.5 d, P=0.0612) (FIG. 5). Bortezomib therapycombined with IMMU-114 or IMMU-140 did not improve survival abovemonotherapy (FIG. 5).

Mice bearing JVM-3 CLL xenografts (FIG. 6) demonstrated similarsensitivity to both IMMU-140 and IMMU-114. There was a significantsurvival benefit in mice treated with either the high (500 mg) or low(100 mg) dose of IMMU-140 vs. saline or control ADC treated mice(P<0.0002) (FIG. 6). Likewise, mice treated with either dose of IMMU-114had >96-d MSTs (P<0.0001 vs. saline and P<0.0003 vs. control ADC) (FIG.6). There are no significant differences between mice treated withIMMU-140 and IMMU-114 at the doses administered to the mice (FIG. 6).These results demonstrate that efficacy may be achieved at doses lessthan 100 mg which suggest a wide therapeutic window clinically forIMMU-140 in this disease.

In all experiments, therapy with IMMU-140 was well tolerated, asevidenced by no significant loss in body weight

Conclusions

IMMU-114 is an IgG₄ Mab that lacks immune functions, eliminating knownadverse events of prior HLA-DR Mabs. HLA-DR, as recognized by IMMU-114,is expressed on a wide range of human hematopoietic and solid cancertypes. Conjugating 6-8 SN-38 molecules via a cleavable linker toIMMU-114 (hL243-SN-38) did not alter its binding to HLA-DR positivecells.

hL243-SN-38 (IMMU-140) provides an added benefit of a dual-therapeuticthrough the direct antitumor activity mediated by the IMMU-114HLA-DR-binding moiety (p-ERK1/2 and AIF signaling) and the addedcytotoxic effect of SN-38 delivery to the cells (caspase cascade andPARP cleavage). IMMU-140 antibody-drug conjugate showed higher potencythan naked IMMU-114 pre-clinically in ALL and AML and an added, if notsignificant, survival benefit in experimental MM and CLL. Overall, thedual-therapeutic potential of SN-38-conjugated IMMU-114 (IMMU-140)allows for the ability to treat a range of HLA-DR-positive hematopoieticand solid cancers.

Therapy with the IMMU-140 ADC proved to be superior to IMMU-114 (whichis active clinically in NHL and CLL) in both AML and ALL xenografts, andbeneficial in MM and CLL. Most importantly, in IMMU-114-refractive AML,IMMU-140 demonstrated a significant antitumor effect without any unduetoxicity. The data show that this new ADC is of use in these intractablemalignancies.

Example 2. Efficacy of IMMU-140 in HLA-DR⁺ Human Melanoma

Expression of the HLA-DR antigen is not limited to hematopoietic cancer,but rather is also found in skin, esophageal, stomach, colon, rectal,pancreatic, lung, breast, ovarian, bladder, endometrial, cervical,testicular, melanoma, kidney, and liver cancers. The present study wasconducted to examine the efficacy of IMMU-140 in non-hematopoieticHLA-DR⁺ tumors.

Cell Binding Studies

LUMIGLO® chemiluminescent substrate system was used to detect antibodybinding to cells. Briefly, A-375 human melanoma cells were plated into a96 black-well, flat-clear-bottom plate overnight. The hL243-γ4P antibodywas added to triplicate wells (2 μg/mL final concentration in the well).As a control for non-specific binding, a humanized anti-CD22 antibodywas likewise added to another set of triplicate wells. Two plates wereset up in which one was incubated at room temperature (RT) and one at 4°C. After incubating for 1 h the media was removed and the cells washedwith fresh, cold media followed by the addition of a 1:20,000 dilutionof goat-anti-human horseradish peroxidase-conjugated secondary antibodyfor 1 h at 4° C. The plates were again washed before the addition of theLUMIGLO® reagent.

Plates were read for luminescence using an ENVISION™ plate reader. Meanluminescent values were determined and graphed as shown in FIG. 7. Meanluminescence of hL243γ4 on A-375 cells, at both 4° C. and RT, wasgreater than 48-fold higher than background and 25-fold higher thannon-specific control, consistent with high expression of HLA-DR on thishuman melanoma cell line. In all, 4 of 4 human melanoma cell linestested (A-375, SK-MEL-28, SK-MEL-5, and SK-MEL-2) were positive forhL243-γ4P binding (48-, 25-, 12-, and 2-fold above background,respectively).

In Vivo Efficacy in A-375 Tumor Xenografts.

Athymic NCr nu/nu nude mice were injected s.c. with 5×10⁶ A-375 cellsper mouse. Once tumors reached approximately 0.3 cm³ in size, theanimals were divided into six different treatment groups of 10 miceeach. Mice received 250 μg i.p. injections of IMMU-140 (hL243-SN-38)(DAR=5.05) twice a week for four weeks. An ADC control group consistedof mice receiving the same doses of non-tumor targeting anti-CD20 ADC(hA20-CL2A-SN-38; DAR=6.08) on the same schedule. Additionally, onegroup of mice received naked hL243-γ4P alone (250 μg) and one grouphL243-γ4P plus irinotecan at doses equivalent to the ADC dose (250 μgMAb+7.5 μg irinotecan). A final group received only irinotecan at10-fold higher doses than the amount of SN-38 carried by hL243-SN-38(i.e., 75 μg). All irinotecan injections were administered as i.v.injections. A final group of mice received only saline (100 μL i.p.).Treatment groups are summarized in Table 4 below.

TABLE 4 Treatment Groups for Melanoma-Bearing Nude Mice hL243-CL2A-SN-38Therapy of Mice Bearing Human Melanoma Tumors (A-375) Group (N) AmountInjected Schedule 1 10 Saline Twice weekly x 4 wks (100 μL i.p.) 2 10hL243-CL2A-SN-38 Twice weekly x 4 wks (250 μg i.p.) 3 10 hA20-CL2A-SN-38Twice weekly x 4 wks (250 μg i.p.) 4 10 hL243-γ4P + Irinotecan Twiceweekly x 4 wks (250 μg i.p. + 7.5 μg i.v.) 5 10 hL243-γ4P Alone Twiceweekly x 4 wks (250 μg i.p.) 6 10 Irinotecan Alone Twice weekly x 4 wks10-fold excess (75 μg i.v.)

Tumors were measured and mice weighed weekly. Animals were euthanizedfor disease progression if their tumor volume exceeded 2.0 cm³ in size.A partial response was defined as shrinking the tumor >30% from initialsize. Stable disease was when the tumor volume remains between 70% and120% of initial size. Time-to-tumor progression (TTP) was determined astime when tumor grew more than 20% from its nadir.

Statistical analysis for the tumor growth data was based on area underthe curve (AUC) and TTP. Profiles of individual tumor growth wereobtained through linear curve modeling. An F-test was employed todetermine equality of variance between groups prior to statisticalanalysis of growth curves. A two-tailed t-test was used to assessstatistical significance between all the various treatment groups andcontrols except for the saline control in which a one-tailed t-test wasused in the analysis. As a consequence of incompleteness of some of thegrowth curves (due to deaths), statistical comparisons of AUC was onlyperformed up to the time at which the first animal within a group wassacrificed. A two-tailed t-test was used to compare TTP values betweengroups.

Mean tumor volume for all the groups when therapy began was 0.314±0.078cm³. Mean tumor growth curves are shown in FIG. 8. This disease modelproved to be very aggressive with saline control tumors progressingrapidly (TTP=7 days; Table 5). While tumors likewise progressed in thecontrol groups, all treatments were able to slow tumor growth relativeto saline control (P<0.0142, AUC) (FIG. 8 and Table 5). However, onlymice treated with hL243-SN-38 demonstrated a significant antitumoreffect when compared to all other groups (P<0.0244; AUC) (FIG. 8 andTable 5). All the mice in this group were partial responders with twomice tumor-free when the experiment ended on therapy day 70. Thisresulted in a greater than 3-fold delay in tumor progression whencompared to all the other non-ADC control groups (P<0.0005) (FIG. 8 andTable 5). Even though this tumor was sensitive to the non-specific ADC,treatment with hL243-SN-38 imparted an 80% greater delay in TTP comparedto mice treated with the control ADC (28±9.9 days vs. 15.6±7.7 days,respectively; P=0.012) (FIG. 8 and Table 5). These data demonstrate thateven in a mouse disease-model of an aggressive human melanoma tumor,therapy with hL243-SN-38 resulted in significant tumor regressions anddelay in disease progression (FIG. 8 and Table 5).

TABLE 5 Time-to-tumor progression for A-375 tumor- bearing mice treatedwith hL243-SN-38. hL243-SN-38 vs. % PR TTP Controls Treatment N (TF)(days) (P-value) hL243-SN-38 10 100  28.0 ± 9.9  N.A. (2) Control ADC 1030  15.6 ± 7.0  0.0120 (1) CTP-11 10 0 8.4 ± 4.4 0.0005 (0) NakedhL243 + 10 0 7.0 ± 0.0 0.0005 CPT-11 (0) Naked hL243 10 0 7.0 ± 0.00.0005 (0) Saline  9* 0 7.0 ± 0.0 0.0003 (0) N = Number of mice pergroup *One mouse censored as an outlier (Critical-Z test). % PR =Percent of mice that exhibited a positive response to treatment TF =Number of mice tumor-free when experiment ended. TTP = Time to TumorProgression for mice not tumor-free N.A. = Not Applicable

Example 3. Preparation of CL2A-SN-38

To the mixture of commercially available Fmoc-Lys(MMT)-OH (0.943 g),p-aminobenzyl alcohol (0.190 g) in methylene chloride (10 mL) was addedEEDQ (0.382 g) at room temperature and stirred for 4 h. Extractive workup followed by flash chromatograph yielded 1.051 g of material as whitefoam. All HPLC analyses were performed by Method B as stated in‘General’ in section 0148. HPLC ret. time: 3.53 min., Electrospray massspectrum showed peaks at m/e 745.8 (M+H) and m/e 780.3 (M+CI),consistent with structure. This intermediate (0.93 g) was dissolved indiethylamine (10 mL) and stirred for 2 h. After solvent removal, theresidue was washed in hexane to obtain 0.6 g of the intermediate ((2) inScheme-3) as colorless precipitate (91.6% pure by HPLC). HPLC ret. time:2.06 min. Electrospray mass spectrum showed peaks at m/e 523.8 (M+H),m/e 546.2 (M+Na) and m/e 522.5 (M−H).

This crude intermediate (0.565 g) was coupled with commerciallyavailableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(‘PEG-N3’, 0.627 g) using EEDQ in methylene chloride (10 mL). Solventremoval and flash chromatography yielded 0.99 g of the product ((3) inScheme-3; light yellow oil; 87% yield). HPLC ret. time: 2.45 min.Electrospray mass spectrum showed peaks at m/e 1061.3 (M+H), m/e 1082.7(M+Na) and m/e 1058.8 (M−H), consistent with structure. Thisintermediate (0.92 g) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate in methylene chloride (10 mL) for 10min under argon. The mixture was purified by flash chromatography toobtain 0.944 g as light yellow oil ((6) in Scheme-3; yield=68%). HPLCret. time: 4.18 min. To this intermediate (0.94 g) in methylene chloride(10 mL) was added the mixture of TBAF (1M in THF, 0.885 mL) and aceticacid (0.085 mL) in methylene chloride (3 mL), then stirred for 10 min.The mixture was diluted with methylene chloride (100 mL), washed with0.25 M sodium citrate and brine. The solvent removal yielded 0.835 g ofyellow oily product. HPLC ret. time: 2.80 min., (99% purity).Electrospray mass spectrum showed peaks at m/e 1478 (M+H), m/e 1500.6(M+Na), m/e 1476.5 (M−H), m/e 1590.5 (M+TFA), consistent with structure.

This azido-derivatized SN-38 intermediate (0.803 g) was reacted with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.233 g)in methylene chloride (10 mL) in presence of CuBr (0.0083 g,), DIEA(0.01 mL) and triphenylphosphine (0.015 g), for 18 h. Extractive workup, including washing with and 0.1M EDTA (10 mL), and flashchromatography yielded 0.891 g as yellow foam. (yield=93%), HPLC ret.time: 2.60 min. Electrospray mass spectrum showed peaks at m/e 1753.3(M+H), m/e 1751.6 (M−H), 1864.5 (M+TFA), consistent with structure.Finally, deprotection of the penultimate intermediate (0.22 g) with amixture of dichloroacetic acid (0.3 mL) and anisole (0.03 mL) inmethylene chloride (3 mL), followed by precipitation with ether yielded0.18 g (97% yield) of CL2A-SN-38; (7) in Scheme-3) as light yellowpowder. HPLC ret. time: 1.88 min. Electrospray mass spectrum showedpeaks at m/e 1480.7 (M+H), 1478.5 (M−H), consistent with structure.

Example 4. Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

Each antibody was reduced with dithiothreitol (DTT), used in a50-to-70-fold molar excess, in 40 mM PBS, pH 7.4, containing 5.4 mMEDTA, at 37° C. (bath) for 45 min. The reduced product was purified bysize-exclusion chromatography and/or diafiltration, and wasbuffer-exchanged into a suitable buffer at pH 6.5. The thiol content wasdetermined by Ellman's assay, and was in the 6.5-to-8.5 SH/IgG range.Alternatively, the antibodies were reduced with Tris (2-carboxyethyl)phosphine (TCEP) in phosphate buffer at pH in the range of 5-7, followedby in situ conjugation. The reduced MAb was reacted with CL2A-SN-38using DMSO at 7-15% v/v as co-solvent, and incubating for 20 min atambient temperature. The conjugate was purified by centrifuged SEC,passage through a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. This way, theSN-38/MAb substitution ratios were determined. The purified conjugateswere stored as lyophilized formulations in glass vials, capped undervacuum and stored in a −20° C. freezer. SN-38 molar substitution ratios(MSR) obtained for some of these conjugates, which were typically in the5-to-7 range, are shown in Table 6.

TABLE 6 SN-38/MAb Molar substitution ratios (MSR) in some conjugates MAbConjugate MSR hMN-14 hMN-14-[CL2A-SN-38] 6.1 hRS7 hRS7-CL2A-SN-38 usingdrug-linker of Example 10 5.8 hA20 hA20-CL2A-SN-38 using drug-linker ofExample 10 5.8 hLL2 hLL2-CL2A-SN-38 using drug-linker of Example 10 5.7hPAM4 hPAM4-CL2A-SN-38 using drug-linker of Example 10 5.9

Example 5. Use of hL243-SN-38 to Treat Therapy-Refractive MetastaticColonic Cancer (mCRC)

The patient is a 67-year-old man who presents with metastatic coloncancer. Following transverse colectomy shortly after diagnosis, thepatient then receives 4 cycles of FOLFOX chemotherapy in a neoadjuvantsetting prior to partial hepatectomy for removal of metastatic lesionsin the left lobe of the liver. This is followed by an adjuvant FOLFOXregimen for a total of 10 cycles of FOLFOX.

CT shows metastases to liver. His target lesion is a 3.0-cm tumor in theleft lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Baseline CEA is 685 ng/mL.

After the patient signs the informed consent, hL243-SN-38 (10 mg/kg) isgiven every other week for 4 months. The patient experiences nausea(Grade 2) and fatigue (Grade 2) following the first treatment andcontinues the treatment without major adverse events. The first responseassessment done (after 8 doses) shows shrinkage of the target lesion by26% by computed tomography (CT) and his CEA level decreases to 245ng/mL. In the second response assessment (after 12 doses), the targetlesion has shrunk by 35%. His overall health and clinical symptoms areconsiderably improved.

Example 6. Treatment of Relapsed Follicular Lymphoma with IMMU-140(Anti-HLA-DR-SN-38)

After receiving R-CHOP chemotherapy for follicular lymphoma presentingwith extensive disease in various regional lymph nodes (cervical,axillary, mediastinal, inguinal, abdominal) and marrow involvement, this68-year-old man is given the experimental agent, IMMU-114-SN-38(anti-HLA-DR-SN-38) at a dose of 10 mg/kg weekly for 3 weeks, with arest of 3 weeks, and then a second course for another 3 weeks. He isthen evaluated for change in index tumor lesions by CT, and shows a 23%reduction according CHESON criteria. The therapy is repeated for another2 courses, which then shows a 55% reduction of tumor by CT, which is apartial response.

Example 7. Treatment of Relapsed Chronic Lymphatic Leukemia withIMMU-140

A 67-year-old man with a history of CLL, as defined by the InternationalWorkshop on Chronic Lymphocytic Leukemia and World Health Organizationclassifications, presents with relapsed disease after prior therapieswith fludarabine, dexamethasone, and rituximab, as well as a regimen ofCVP. He now has fever and night sweats associated with generalized lymphnode enlargement, a reduced hemoglobin and platelet production, as wellas a rapidly rising leukocyte count. His LDH is elevated and thebeta-2-microglobulin is almost twice normal. The patient is giventherapy with IMMU-114-SN-38 conjugate at a dosing scheme of 8 mg/kgweekly for 4 weeks, a rest of 2 weeks, and then the cycle repeatedagain. Evaluation shows that the patient's hematological parameters areimproving and his circulating CLL cells appear to be decreasing innumber. The therapy is then resumed for another 3 cycles, after whichhis hematological and lab values indicate that he has a partialresponse.

Example 8. Immunoconjugate Storage

The conjugates described in above were purified and buffer-exchangedwith 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, and furtherformulated with trehalose (25 mM final concentration) and polysorbate 80(0.01% v/v final concentration), with the final buffer concentrationbecoming 22.25 mM as a result of excipient addition. The formulatedconjugates were lyophilized and stored in sealed vials, with storage at2° C.-8° C. The lyophilized immunoconjugates were stable under thestorage conditions and maintained their physiological activities.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

We claim:
 1. A method of treating an HLA-DR⁺ cancer comprisingadministering to a human patient with an HLA-DR⁺ acute myeloid leukemia,an immunoconjugate IMMU-140.
 2. The method of claim 1, wherein theHLA-DR⁺ cancer does not respond to treatment with unconjugatedanti-HLA-DR antibody.
 3. The method of claim 1, wherein theimmunoconjugate is administered as a front-line therapy to patients whohave not previously been treated for the cancer.
 4. The method of claim1, wherein the immunoconjugate is administered to a patient who haspreviously relapsed from or been resistant to at least one anti-cancertherapy.
 5. The method of claim 1, wherein the immunoconjugate isadministered to a patient who is not eligible for stem-cell orbone-marrow transplantation.
 6. The method of claim 1, wherein theimmunoconjugate is administered at a dosage of between 3 mg/kg and 18mg/kg.
 7. The method of claim 6, wherein the dosage is selected from thegroup consisting of 4 mg/kg, 6 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12mg/kg, 16 mg/kg and 18 mg/kg.
 8. The method of claim 1, wherein theimmunoconjugate is administered at a dosage of between 6 mg/kg and 12mg/kg.
 9. The method of claim 1, wherein the immunoconjugate isadministered at a dosage of between 8 mg/kg and 10 mg/kg.
 10. The methodof claim 1, wherein the cancer is metastatic.
 11. The method of claim10, further comprising reducing in size or eliminating the metastases.12. The method of claim 4, wherein the patient has failed to respond totherapy with irinotecan, prior to treatment with the immunoconjugate.13. The method of claim 6, wherein the immunoconjugate dosage isadministered to the human patient once or twice a week on a schedulewith a cycle selected from the group consisting of: (i) weekly; (ii)every other week; (iii) one week of therapy followed by two, three orfour weeks off; (iv) two weeks of therapy followed by one, two, three orfour weeks off; (v) three weeks of therapy followed by one, two, three,four or five weeks off; (vi) four weeks of therapy followed by one, two,three, four or five weeks off; (vii) five weeks of therapy followed byone, two, three, four or five weeks off; and (viii) monthly.
 14. Themethod of claim 13, wherein the cycle is repeated 4, 6, 8, 10, 12, 16 or20 times.
 15. The method of claim 1, wherein the immunoconjugate isadministered in combination with one or more therapeutic modalitiesselected from the group consisting of unconjugated antibodies,radiolabeled antibodies, drug-conjugated antibodies, toxin-conjugatedantibodies, gene therapy, chemotherapy, therapeutic peptides, cytokinetherapy, oligonucleotides, localized radiation therapy, surgery andinterference RNA therapy.
 16. The method of claim 15, wherein thetherapeutic modality comprises treatment with a therapeutic agentselected from the group consisting of 5-fluorouracil, afatinib, aplidin,azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291,bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan,calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin,carmustine, celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors,irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans,cyclophosphamide, crizotinib, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, entinostat, estrogen receptor bindingagents, etoposide (VP16), etoposide glucuronide, etoposide phosphate,exemestane, fingolimod, flavopiridol, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, fostamatinib, ganetespib,GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib,idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin,SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC),transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839.
 17. The method of claim 15,wherein the immunoconjugate is administered in combination with a secondantibody or antigen-binding fragment thereof that binds to atumor-associated antigen (TAA) selected from the group consisting ofcarbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, ART-4, Ba733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21,CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67,CD70, CD7OL, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138,CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12,HIF-1α, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met,DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblastgrowth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE,gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG),HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia,IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23,IL-25, insulin-like growth factor-1 (IGF-1), KS1-4, Le-Y, LDR/FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,MART-2, NY-ESO-1, TRAG-3, CRP, MCP-1, MIP-1A, MIP-1B, MUC1, MUC2, MUC3,MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,pancreatic cancer mucin, PD-1 receptor, placental growth factor, p53,PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF,ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin,survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factor C3, C3a, C3b, C5a,C5, an angiogenesis marker, bc1-2, bc1-6, Kras, and an oncogene product.18. The method of claim 17, wherein the TAA is selected from the groupconsisting of CD19, CD20, CD22 and CD74.
 19. The method of claim 17,wherein the second antibody is selected from the group consisting ofhA19, hA20, hLL1 and hLL2.
 20. The method of claim 1, wherein theimmunoconjugate is administered subcutaneously.
 21. The method of claim20, wherein the immunoconjugate is administered at a dosage of 2 to 4mg/kg.
 22. The method of claim 20, wherein the immunoconjugate isadministered in a volume of 1, 2 or 3 ml or less.
 23. The method ofclaim 20, wherein a maintenance dose of the immunoconjugate isadministered subcutaneously after the immunoconjugate is administeredintravenously to the same subject.